EXTREMOPHILIC BACTERIA in the HIGH-ALTITUDE AQUATIC HABITATS of OJOS DEL SALADO VOLCANO Phd Thesis

Home , Laguna Verde (volcano), Socompa

EÖTVÖS LORÁND UNIVERSITY Faculty of Science Department of Microbiology

Júlia Margit Aszalós

EXTREMOPHILIC BACTERIA IN THE HIGH-ALTITUDE AQUATIC HABITATS OF OJOS DEL SALADO VOLCANO PhD thesis

Supervisor: Dr. Andrea Borsodi habilitated associate professor

Doctoral School of Biology Head of doctoral school: Dr. Anna Erdei

Zootaxonomy, Animal Ecology, Hydrobiology PhD Program Head of PhD program: Dr. János Török

Budapest, 2019

1 Introduction and aims

• The Dry-Andes located in the central part of the Andes extends 1500 km from north to south, harboring one of the highest altitude deserts of Earth, the Puna de Atacama plateau with an average altitude of 3500 m above sea level. In this area, several volcanoes are found, along with Ojos del Salado (6893 m), the highest active volcano on Earth. • The region is characterized by high-altitude desert climate with an average annual precipitation of less than 150 mm, low mean temperature, extreme daily temperature range extending from 20°C to +30°C and permafrost above 5600 m a.s.l. At this altitude, UV radiation is more intense than at the sea level, while the volcanism present in the area contributes to the presence of warm springs, fumaroles often with strongly acidic water (Nagy et al., 2018). The unique mountain desert climate, the high altitude and the volcanism together contributes to the extremity of habitats in this region. • Highland desert wetlands are considered as extreme environments where low productivity communities form simplified foodwebs. The taxon diversity of the bacterial communities and the abundance of each species are influenced by the altitude, temperature and salt concentration (Cabrol et al., 2009). In such extreme conditions extremophilic microorganisms, mostly prokaryotes are present. • This research focused on bacterial communities harbored by two salt lakes of the Puna de Atacama plateau, Laguna Santa Rosa (3770 m a.s.l.) and Laguna Verde (4350 m a.s.l.) along with two lakes of Ojos del Salado volcano: a permafrost thaw pond at 5900 m a.s.l. and a crater lake at 6480 m a.sl., where acidic warm water was present due to the fumarolic activity. The diversity of bacterial communities in these environments has not yet been studied by cultivation or next generation sequencing methods. • The aim of our study was to examine the phylogenetic diversity of bacterial communities harbored by the aquatic habitats in the region of Ojos del Salado using culture and culture- independent molecular biological methods. Our goal was to test the ecological tolerance of the isolated bacterial strains and compare the NaCl tolerance of bacterial strains isolated from the two high-altitude salt lakes, as well as the pH tolerance of strains from the sites above the permafrost limit. Spatial heterogeneity of bacterial communities in the permafrost thaw pond was also explored by comparing the diversity of permafrost and lake sediments from different depths, as well as the diversity of aquatic bacterial communities. In addition,

the bacterial diversity of cold and warm water sediments in the aquatic environments of the crater was also contrasted.

2 Materials and methods

• Samplings took place during the expedition of the Hungarian Dry-Andes Research Program in February 2016. Samples were collected from altogether 4 sampling sites; the type and origin of the samples are listed in Table 1. Sediment samples were obtained using a sterile spatula into 50 ml Falcon tubes, water samples were collected into sterilized plastic bottles. • Cultivation of sediment samples was performed using R2A and PYG media, numbers of colony forming units were estimated and bacterial isolates were obtained. To identify the bacterial strains, DNA was extracted by disrupting the cells with glass-betaing method and 16S rRNS gene was amplified using PCR with bacteria specific primers (27F–1401R). Bacterial isolates were grouped with ARDRA method (Amplified Ribosomal DNA Restriction Analysis). 16S rRNA gene sequence of at least one strain from each ARDRA group was determined and identified according to the NCBI database. Bacterial strains derived from the salt lakes were tested for NaCl tolerance in nutrient broths containing 0, 2.5, 5.0, 7.5 and 10.0% of NaCl. Bacterial strains from the permafrost thaw pond and the crater lake sediment were tested for pH tolerance in nutrient broths adjusted to pH 2, 3, 4, 5, 6 and 7. In te case of both tests optical density of the inoculated broths was measured after 2 weeks of incubation. • To assess the diversity of bacterial communities with molecular methods, community DNA was extracted from the samples using the QIAGEN DNEasy Power Soil kit according to the manufacturer’s instructions. The structure of bacterial communities in each sample was compared using DGGE (denaturing gradient gel electrophoresis) by separating 16S rRNA gene PCR amplicons obtained with a semi-nested PCR in a 7% polyacrylamide gel containing 40-60% denaturing gradient. The TotalLab sotfware was used to compare molecular fingerprints of the samples. Based on the results of DGGE, samples were chosen for an in-depth analysis of bacterial communities with next-generation sequencing. Amplicon libraries were generated by PCR amplification of the V3-V4 region of the 16S rRNA gene using B341F and B805NR primers from the community DNA isolates. Samples obtained from the salt lakes were sequenced using the Ion Personal Genome MachineTM (PGMTM) according to the manufacturer’s instructions with the Ion PGMTM Hi-QTM View

Sequencing Kit using the Ion 314TM Chip v2 BC at the Department of Microbiology, ELTE. To sequence the samples from the thaw pond and the crater lake on the Illumina MiSeq platform, the services of the Research Technology Support Facility, Genomics Core Lab of the Michigan State University (USA) were used. The bioinformatical analysis of the obtained sequencing datasets was done using the mothur v1.38.1 software according to the 454 or the MiSeq SOP. • 1. Table. Samples collected from aquatic habitats of the Puna de Atacama plateau and Ojos del Salado, and corresponding methods (C: cultivation, D: DGGE, S: sequencing; samples collected for water chemistry are signed with *)

Sample Sample type C D S identifier SR-S1 Lake shore sediment x x x SR-S2 Lake shore sediment x x x SR-S3 Clay sediment x x x

3770 m 3770 SR-S4 Clay sediment x x x

L.Rosa, Santa SR-W* Lake water LV-S1 Warm lagoon sediment x x x LV-S2 Warm lagoon mat x x x LV-S3 Warm lagoon mat x x x LV-S4 Warm lagoon mat x x x LV-P Lake water x

L. Verde, 4350 m 4350 L.Verde, LV-W* Lake water , , S7 Lake sediment x x x S8 Lake sediment x x x

S9 Permafrost sediment x x x

thaw pond thaw S10 Permafrost sediment x x x S11 Permafrost sediment x x x 5900 m 5900 S12 Permafrost sediment x x x F Lake water x

Permafrost Permafrost TO-W* Lake water Fumarolic creek , , x x FC-1 sediment Fumarolic creek x x

FC-2 sediment aterlake

6480 m 6480 LS Lake sediment x x Cr KR-F* Lake water

3 Results and discussion

• Cultivation based and molecular methods were used to reveal the phylogenetic diversity of bacterial communities haboured by the high-altitude aquatic environments of the Puna de Atacama plateau and Ojos del Salado volcano. • The bacterial communities in the sediment of Laguna Santa Rosa, the microbial mats of Laguna Verde, the water and sediment of a permafrost thaw pond and the surrounding permafrost sediment, as well as the sediments of a lake and a fumarolic creek in the Ojos del Salado crater were analysed. On site measurements showed that physical-chemical properties (e.g. pH, temperature, conductivity) of the examined environments varied on a broad scale. Both salt lakes had conductivity over 5 mS/cm, but their temperature and pH were different (Laguna Santa Rosa: 18.8 °C and pH 8.1; Laguna Verde: 38.8 °C and 6.8). Temperature and pH of water in the permafrost thaw pond was 9.2 °C and 8.7, respectively. In the volcanic crater, fumarolic creek water was acidic (pH 2.1) and its temperature decreased from 40.8°C at 20 m from the source to 34.7°C at 50 m from the source. Temperature and pH in the crater lake water was 3.8°C and 4.9, respectively. • The cultivation was performed on R2A and PYG media. A higher number of colony forming units (CFUg-1) and cultivable diversity were obtained on the oligotrophic R2A, than on the PYG plates. Although PYG was formerly applied for cultivation from similar environments, R2A (probably due to its lower organic matter content) proved to be more suitable for cultivation. • Bacterial strains isolated from the Laguna Santa Rosa affiliated with members of phyla Proteobacteria, Bacteroidetes and Firmicutes. From the Laguna Verde and the crater lake, members of phyla Proteobacteria and Actinobacteria were identified, however, with one exception (Marinobacter guineae) the recognized species were different. From the sediment of the permafrost thaw pond members of phyla Proteobacteria, Actinobacteria and Bacteroidetes were isolated. • Cultivation allowed us to determine ecological tolerance for e.g. pH or NaCl of the strains derived from different environments. NaCl tolerance tests of strains isolated from the salt lakes showed that bacteria from Laguna Santa Rosa preferred higher salt concentrations (7.0-10.0% NaCl). Contrary to this, Laguna Verde strains showed a moderate salt tolerance, growing only at 2.5-5.0% NaCl. Based on these results, bacterial strains derived from the constantly salty environment of Laguna Santa Rosa are better adapted to NaCl

concentrations than those from Laguna Verde, where inflows from warm springs might contribute to a more variable environment regarding salt concentrations, as well. During the cultivation, it was complicated to maintain bacterial strains from microbial mats of Laguna Verde, which might be the effect of the complex syntrophic relationships between bacteria within biofilms. Strains were unable to grow after isolation, when these relationships were terminated, since these species could be mutually dependent on each other. • The pH tolerance results of strains from the thaw pond and crater lake revealed that isolates from the acidic sediment of the crater lake showed a broader tolerance towards lower pH values (pH 4) than those from the thaw pond (pH 6). This indicates that fumaroles supported by volcanic activity are having strong impact on the ecological tolerance of bacterial communities through lowering the pH. • Result of the next-generation sequencing revealed high ratio of sequences affiliated with hitherto unknown bacterial species or candidate taxa in these extreme environments. Applying the 97% level of sequence similarity as the limit of species identification, high proportion of sequences belonged to uncultured genera in all of the studied habitats. In the salt lakes most of these genera belonged to phyla Gralicibacteria, Firmicutes and Bacteroidetes. Phylum Parcubacteria was present in every assessed sample, and was outsandingly dominant in a microbial mat sample of Laguna Verde. At least 46% of the sequences detected from the permafrost thaw pond affiliated with cadidate taxa. Some of the most abundand genera also belonged to uncultured or unclassified taxa. Most of these were members of phyla Patescibacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, Proteobacteria or Verrucomicrobia. Unknown bacterial species were also present in the sediment of the crater lake and the fumarolic creek. Altogether six candidate phyla, superphyla or division (Dependentia, Rokubacteria, Gracilibacteria, Microgenomatia, Parcubactera, Patescibacteri and WPS-2) were deteceted. The relative abundance of candidate division WPS-2 was outstanding in the warm water saturated fumarolic creek sediment. These results are in line with other studies focusing on bacterial communities in aquatic habitats of the Dry-Andes. It supports the idea that remote high-altitude aquatic habitats are local biodiversity hotspots harbouring hitherto unknown microorganisms. Since extremophilic bacteria has a broad potential for application in various fields of industry and biotechnology, these results draw attention to the importance of studying bacterial communities in such environments. • Molecular methods revealed simplified bacterial communities with a low taxonomic diversity in all of the studied habitats. The results of the cultivation and molecular methods 6

revealed the presence of extremophilic or polyextremophilic bacteria in the studied aquatic environments. In the high-altitude salt lakes of the Puna de Atacama-plateau halophilic and halotolerant bacteria were detected. In the habitats of the permafrost thaw pond mainly psychrophilic or psychrotolerant bacteria were detected, belonging to species frequently present in oligotrophic environments. In the acidic sediments of the volcanic crater, acidophilic or psychrophilic taxa were also identified. Both cultivation and sequencing showed that the composition of bacterial communities are shaped by the characteristics of mountain desert climate (e.g. great daily temperature range, aridity), effects of high-altitude (e.g. intense UV radiation, low mean temperature) and features attributable to volcanic activity (i.e. presence of fumaroles and low pH). • In the Laguna Santa Rosa sediments and Laguna Verde microbial mats the highest abundance of phyla Proteobacteria and Bacteroidetes was detected, both of them are ferquently present in saline habitats (Oren, 2008). Members of Firmicutes were also abundant, as well as Cyanobacteria, Parcubacteria and Chloroflexi, in the microbial mat of Laguna Verde. The revealed bacterial diversity was similar to those formerly described from other high-altitude saline habitats of the Dry-Andes (e.g. Salar de Coposa, Salar de Puja), or from high-altitude lakes of the Tibetan plateau (Rasuk et al., 2014, Yang et al., 2016). Although regarding the detected phyla several similarities were discovered, on lower taxonomic levels characteristic differences were found, e.g. dominant genera differed between environments on different geographical locations. • Bacterial communities in the permafrost thaw pond of Ojos del Salado were consisting of taxa (e.g. Proteobacteria, Bacteroidetes, Actinobacteria) characteristic to different types of periglacial environments. Structure of bacterial communities in the water, the lake sediments and in the surface and subsurface permafrost sediments showed great variance forming four distinct groups on the principal component analysis plot. Based on these results, the main differences between bacterial communities in the sediments were attributable to limiting factors like intense radiation, regular freeze-thaw cycles and regular desiccation. The revealed taxonomic diversity was similar to those detected on other volcanos in the Dry- Andes, e.g. Socompa or Llullaillaco, and in degrading Arctic permafrosts like those in Nunavut, Canada (Solon et al., 2018, Steven et al., 2007). • In the habitats of the crater, representatives of phyla Proteobacteria and Actinobacteria were the most abundant, while members of Bacteroidetes, contrary to the environments of the thaw pond were sparse. Bacterial communities in the sediments saturated with cold and warm water were characteristically different, in the fumarolic creek sediments phylotypes 7

belonging to Aquificae, WPS-2, Firmicutes or Chloroflexi were abundant, while in the lake sediment Patesibacteria formed a major group. These differences also appeared on lower taxonomic levels: in the warm environments genera Acidithrix, Ferrithrix, Hydrogenobaculum, Acidibacter, Metallibacterium, Gallionella, Thiomonas and Acidiphilum were detected, while others, e.g. Granulicella, Sulfuriferula and Rhodanobacter were revealed mainly from cold sediment. Bacterial communities in the sediment of the crater lake were similar to those in cryoconites of polar ice-sheets in Greenland, and permafrost sediments of other volcanoes (Lluillaillaco, Socompa) in the Dry-Andes (Solon és mtsai, 2018, Cameron és mtsai, 2015). The fumarolic creek sediment harboured communities similar to those in other acidic environments, e.g. permafrost of the Antarctic Mitchell peninsula or microbial mats around hot springs in the Yellowstone National Park, USA (Ji et al., 2016, Donahoe-Christiansen és mtsai, 2004). • Based on our results, it was also found that taxon diversity decreased by the altitude and the extremity of the habitats. In the sediment of the thaw pond at 5900 m a.s.l. the number of the observed OTUs was five times higher than in the crater lake or the fumarolic creek sediment. However, these resuts were not entirely attributable to the altitude because other physical-chemical properties of the studied habitats (e.g. pH) might also play an important role in shaping bacterial communities. In the salt lakes, the low water activity and the intense UV radiation are the main limiting factors, whereas in the environments of the permafrost thaw pond (besides radiation) the low mean temperature, the regular freeze-thew cycles and the desiccation of the permafrost sediment surface had the strongest impact on community structure. In the crater lake and fumarolic creek the low pH was an additional extreme environmental feature. Based on the next-generation sequencing data, the structure of bacterial communities was shaped by selective forces characteristic to each environment, therefore phylogenetic and potential metabolic diversity of bacteria differed distinctively in each aquatic habitat. This result was in line with characteristically different ecological tolerance for pH or NaCl of the isolated strains in the studied environments. • Based on the revealed taxon diversity and literature data, the potential metabolic diversity of the studied bacterial communities was manifold. In the salt lakes bacteria capable of 2+ oxidizing thiosulphate, Fe , H2, reducing nitrate or sulfate and perform nitrogen fixation or methylotrophy were detected. In the microbial mats of the warm lagoon of Laguna Verde a high proportion of bacterial species known to participate in the formation of biofilms as primary colonizers were also detected, e.g. members of Rhodobacterales and Arcobacter. In the sediments of Laguna Santa Rosa, the relative abundance of anaerobic species was higher 8

than in Laguna Verde and among the autotrophic bacteria mainly chemolithoautotrophs were detected. Contrary to this, the Laguna Verde microbial mats harboured a more abundant assemblage of phototrophic taxa. Sediments in the environment of the thaw pond harboured simplified bacterial communities with photoautotrophic organisms being the most abundant group of autotrophs, therefore these can be considered as the main primary producers. Despite the low amounts of organic material in these environments, heterotrophic bacteria were diverse, as well. In the crater environments chemolitoautotrophic, fotoautotrophic and heterotrophic bacteria were present. Among them iron-reducers, sulphate-reducers, iron-oxidizers and sulfur-oxidizers were detected. • Revealing of the taxonomic diversity of bacterial communities in the aquatic habitats of Ojos del Salado volcano allowed us to set up four integrated geomicrobiological models. These models show how bacterial taxa frequent and abundant in a given environment participate in the carbon, sulfur, nitrogen and iron biogeochemical cycles.

4 Thesis

• Bacterial diversity of microbial mats and lake sediments in two high-altitude saline lakes, Laguna Santa Rosa and Laguna Verde was revealed with cultivation and molecular methods. We revealed the presence of phyla frequent in saline lakes in both the sediments and microbial mats of the studied environments. Our results showed that besides the ratio of dominant phyla, presence of minor taxa also contributed to the variance among communities in the sediments and microbial mats of the studied lakes. Ecological tolerance of bacterial strains for higher NaCl concentrations revealed that the isolated strains were moderately halophilic. The revealed taxon diversity showed that the Laguna Santa Rosa sediments harboured diverse communities of anaerobic genera and many chemolithoautotrophs, while in the Laguna Verde microbial mats phototrophic bacteria were abundant. • Assessing the culturable diversity of the permafrost thaw pond at 5900 m a.s.l., and tolerance of the isolates towards acidic pH showed the presence of bacterial strains with circumneutral pH optima. Using molecular methods, we revealed the spatial heterogeneity of bacterial communities in and around the pond by comparing bacterial diversity in permafrost and lake sediments, as well as lake water. Results imply that the presence of water and depth of sediment are both responsible for the differences between composition of

bacterial communities in this environment. In the thaw pond sediment mostly aerob or microaerophilic organisms were revealed, many of them characterized by chemolitotrophic metabolism. • In the crater with fumarolic activity at 6480 m a.s.l., a lower taxon diversity was revealed by both cultivation and molecular methods from sediments saturated with cold and warm water than from the other studied sites in the Ojos del Salado region. Pronounced differences between bacterial diversity of the cold lake sediment and warm fumarolic creek sediment showed that volcanism affected bacterial community composition, mainly through pH and temperature. Bacterial strains isolated from the lake sediment showed acidophilic characters with preference towards acidic pH. Regarding metabolic properties, bacterial communities in the crater were characterized by the presence of organisms participating in iron and sulfur biogeochemical cycles.

5 References

Cabrol N.A., Grin E.A., Chong G., Minkley E., Hock A.N., Yu Y., Bebout L., Fleming E., Häder D.P., Demergasso C., Gibson J., Escudero L., Dorador C., Lim D., Woosley C., Morris R.L., Tambley C., Gaete V., Galvez M.E., Smith E., Uskin- Peate I., Salazar C., Dawidowicz G. & Majerowicz J. (2009) The high‐lakes project. J Geophys Res-Biogeo 114(G2) Cameron, K. A., Stibal, M., Zarsky, J. D., Gözdereliler, E., Schostag, M. & Jacobsen, C. S. (2015). Supraglacial bacterial community structures vary across the Greenland ice sheet. FEMS Microbiol Ecol, 92:fiv164 Donahoe-Christiansen, J., D'Imperio, S., Jackson, C. R., Inskeep, W. P., & McDermott, T. R. (2004). Arsenite-oxidizing Hydrogenobaculum strain isolated from an acid- sulfate-chloride geothermal spring in Yellowstone National Park. Appl Environ Microbiol 70:1865-1868. Ji, M., van Dorst, J., Bissett, A., Brown, M. V., Palmer, A. S., Snape, I., Siciliano, S. D. & Ferrari, B. C. (2016). Microbial diversity at Mitchell Peninsula, Eastern Antarctica: a potential biodiversity “hotspot”. Polar Biology, 39:237-249. Nagy, B., Ignéczi, Á., Kovács, J., Szalai, Z. & Mari, L. (2018). Shallow ground temperature measurements on the highest volcano on Earth, Mt. Ojos del Salado, Arid Andes, Chile. Permafrost and Periglacial Processes. Oren, A. (2008). Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline systems, 4:2. Rasuk, M. C., Kurth, D., Flores, M. R., Contreras, M., Novoa, F., Poire, D. & Farias, M. E. (2014). Microbial characterization of microbial ecosystems associated to evaporites domes of gypsum in Salar de Llamara in Atacama desert. Microbial Ecol, 68:483-494. Solon, A. J., Vimercati, L., Darcy, J. L., Arán, P., Porazinska, D., Dorador, C., Farías, M. E & Schmidt, S. K. (2018) Microbial Communities of High-Elevation Fumaroles, Penitentes, and Dry Tephra “Soils” of the Puna de Atacama Volcanic Zone. Microb Ecol 1-12. Steven, B., Briggs, G., McKay, C. P., Pollard, W. H., Greer, C. W. & Whyte, L. G. (2007). Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods. FEMS Microbiology Ecology, 59:513-523. Yang, J., Ma, L. A., Jiang, H., Wu, G. & Dong, H. (2016). Salinity shapes microbial diversity and community structure in surface sediments of the Qinghai-Tibetan Lakes. Scientific reports, 6:25078.

6 Publications in the subject of the PhD thesis

Aszalós J.M., Szabó, A., Megyes, M., Anda, D., Nagy, B., Borsodi, A.K (2019). Bacterial diversity of a high-altitude permafrost thaw pond located on Ojos del Salado (Dry- Andes, Atacama). Astrobiology (under review) Kereszturi, Á., Aszalós, J.M., Heiling, Zs., Kapui, Zs., Király, Cs., Leél-Össy, Sz., Nagy, B., Pál, B., Skulteti, Á., Szalai, Z. (2019). General characteristics of the Ojos del Salado region as a potential Mars analogue site in the Atacama desert. Astrobiology (under review) Aszalós J.M., Szabó, A., Felföldi, T., Jurecska, L., Nagy, B., Borsodi, A.K (2019). Effects of active volcanism on bacterial communities in the highest altitude crater lake of Ojos del Salado (Dry Andes, Atacama). Astrobiology (accepted for publication) Aszalós, J.M., Krett, G., Anda, D., Márialigeti, K., Nagy, B., Borsodi, A.K. (2016). Diversity of extremophilic bacteria in the sediment of high-altitude lakes located in the mountain desert of Ojos del Salado volcano, Dry-Andes. Extremophiles 20: 603-620. • Full publications in conference proceedings: Aszalós, J.M., Krett, G., Anda, D., Márialigeti, K., Nagy, B., Borsodi, A.K. (2016). Extremofil baktériumközösségek az Ojos del Salado vulkán (Száraz-Andok) magashegyi tavainak üledékében. Hidrológiai Közlöny 96: 8-13.

7 Other publications

Borsodi, A.K., Aszalós, J.M., Bihari, P., Nagy, I., Schumann, P., Spröer, C., Kovács, A.L., Bóka, K., Dobosy, P., Óvári, M. Szili-Kovács, T., Tóth E. (2019) Anaerobacillus alkaliphilus sp. nov., a novel alkaliphilic and moderately halophilic bacterium. International Journal of Systematic and Evolutionary Microbiology 69: 631-637. Ujvári, G., Borsodi, A., Aszalós, J.M., Megyes, M., Mucsi, M., Szabó, A., Márialigeti, K. (2018). Mikrobaközösségek metabolikus aktivitása és 16S rRNS gén alapú filogenetikai diverzitása kukorica monokultúra rizoszféra-talajban. Agrokémia és Talajtan, 67:227- 244. Megyes, M., Aszalós, J.M., Móga, J., Márialigeti, K., Borsodi A.K. (2018) A Máramarosi- medence sós tavainak baktériumközösségei. Hidrológiai Közlöny 98: 57-61. Borsodi, A.K., Tóth, E., Aszalós J.M., Bárány Á., Schumann P., Spröer C., Kovács A.L., Márialigeti K., Szili-Kovács T. (2017) Bacillus kiskunsagensis sp. nov., a novel alkaliphilic and moderately halophilic bacterium isolated from soda soil. International Journal of Systematic and Evolutionary Microbiology 67:3490-3495.